Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Here, we performed high-resolution copy-number analysis on 125 HCC tumors and whole-exome sequencing on 24 of these tumors. We identified 135 homozygous deletions and 994 somatic mutations of genes with predicted functional consequences. We found new recurrent alterations in four genes (ARID1A, RPS6KA3, NFE2L2 and IRF2) not previously described in HCC. Functional analyses showed tumor suppressor properties for IRF2, whose inactivation, exclusively found in hepatitis B virus (HBV)-related tumors, led to impaired TP53 function. In contrast, inactivation of chromatin remodelers was frequent and predominant in alcohol-related tumors. Moreover, association of mutations in specific genes (RPS6KA3-AXIN1 and NFE2L2-CTNNB1) suggested that Wnt/β-catenin signaling might cooperate in liver carcinogenesis with both oxidative stress metabolism and Ras/mitogen-activated protein kinase (MAPK) pathways. This study provides insight into the somatic mutational landscape in HCC and identifies interactions between mutations in oncogene and tumor suppressor gene mutations related to specific risk factors.

Radioembolization of liver cancers using 90 Y-loaded microspheres is experiencing more widespread use. However, few data are available concerning the doses delivered to the tumors and the healthy liver. This retrospective study was conducted to calculate the tumor dosimetry (planned tumor dose [T plan D]) and nontumor dosimetry in patients treated by 90 Y-loaded glass microspheres and determine whether tumor dosimetry could predict response and survival. Methods: Thirty-six patients with hepatocellular carcinoma (HCC), including 16 with portal vein thrombosis (PVT), were treated with 90 Y-loaded glass microspheres. The T plan D and the dose delivered to the injected healthy liver were calculated using a quantitative analysis of the 99m Tc-macroaggregated albumin ( 99m Tc-MAA) SPECT/CT exam. Responses were assessed after 3 mo, using the criteria of the European Association for the Study of the Liver. Progression-free survival (PFS) and overall survival (OS) were evaluated using Kaplan-Meier tests. Results: The response rate was 69% for the overall population and 75% for the PVT patients. The dose delivered to the tumor was the only parameter associated with response with multivariate analysis (P 5 0.019). A threshold T plan D value of 205 Gy was predictive of response, with a sensitivity of 100% and an accuracy of 91%. Quantitative 99m Tc-MAA SPECT/CT allowed us to increase the injected activity for 4 patients with large lesions. PFS was only 5.2 mo and OS 9 mo when using a T plan D of less than 205 Gy versus 14 mo (P 5 0.0003) and 18 mo (P 5 0.0322), respectively, with a T plan D of 205 Gy or more. Conclusion: Quantitative 99m Tc-MAA SPECT/ CT is predictive of response, PFS, and OS. Dosimetry based on 99m Tc-MAA SPECT/CT can be used for the selection of patients and for an adaptation of treatment planning, especially in selected patients (particularly in the case of large tumors). These results also confirm the efficacy and safety of 90 Y-loaded microspheres in treating HCC, even in the presence of PVT (and especially when 99m Tc-MAA uptake is seen inside the PVT).

. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT-PCR, cDNA encoding sequences for ADAM9 and ADAM12 were identified in human activated hepatic stellate cells (HSCs). Northern blot analyses showed that HSCs, but not hepatocytes, expressed transcripts for ADAM9 messenger RNA (mRNA) and both the long and short forms of ADAM12. This expression was associated with the transition from quiescent to activated state of rat HSCs and markedly increased in human livers with cirrhosis. ADAM12 but not ADAM9 expression was up-regulated by transforming growth factor ␤ (TGF-␤) in human activated HSCs. The PI3K inhibitor LY294002 and the mitogen-activated protein kinase kinase (MEK) inhibitor UO126 prevented ADAM12 induction by TGF-␤, suggesting the involvement of PI3K and MEK activities. In vivo, the steady-state of both ADAM9 and ADAM12 mRNA levels was nearly undetectable in both normal livers and benign tumors and increased in hepatocellular carcinomas (up to 3-and 6-fold, respectively) and liver metastases from colonic carcinomas (up to 40-and 60-fold, respectively). The up-regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12 expression is associated with tumor aggressiveness and progression. I ncreased expression and activities of matrix metalloproteinase (MMP) has been shown widely in malignant phenotypes facilitating the breakdown of extracellular matrix component and cell evasion, but also unmasking bioactive cryptic fragments and releasing active growth factors which, in turn, favor tumor growth. 1 The ADAMs (a disintegrin and metalloproteinase-containing proteins) are a family of multidomain glycoproteins highly homologous to the class III snake venom metalloprotease-disintegrins. 2 The common extracellular part of the proteins includes a regulatory prodomain and metalloprotease, disintegrin-like and cystein-rich domains. Further, ADAMs are characterized by an epidermal growth factor (EGF)-like domain, a transmembrane domain, and a cytoplasmic tail. More than 30 members have been identified in the ADAM family with a broad tissue distribution and have been involved in specific cellular processes including sperm-egg interaction, 3 myocyte fusion, 4 neurogenesis, 5 and adipogenesis. 6 Recently, ADAM12 gene therapy was shown to rescue the pathology of mdx-gene deficient dystrophic mice.

Many solid malignant tumors arise on a background of inflamed and/or fibrotic tissues, features which are found in more than 80% hepatocellular carcinomas (HCC). Activated hepatic stellate cells (HSC) play a critical role in fibrogenesis associated with HCC onset and progression, yet their functional impact on hepatocyte fate remains largely unexplored. Here, we used a coculture model to investigate the crosstalk between hepatocytes (human hepatoma cells) and activated human HSC. Unsupervised genome-wide expression profiling demonstrated that hepatocyte-HSC crosstalk is bidirectional and results in the deregulation of functionally relevant gene networks. Notably, coculturing increased the expression of pro-inflammatory cytokines and modified the phenotype of hepatocytes toward motile cells. Hepatocyte-HSC crosstalk also generated a permissive pro-angiogenic microenvironment, particularly by inducing VEGFA and MMP9 expression in HSC. An integrative genomic analysis revealed that the expression of genes associated with hepatocyte-HSC crosstalk correlated with HCC progression in mice and was predictive of a poor prognosis and metastasis propensity in human HCC. Interestingly, the effects of crosstalk on migration and angiogenesis were reversed by the histone deacetylase inhibitor trichostatin A. Our findings therefore indicate that the crosstalk between hepatoma cells and activated HSC is an important feature of HCC progression, which may be targeted by epigenetic modulation.

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PurposeTo evaluate the impact of dosimetry based on MAA SPECT/CT for the prediction of response, toxicity and survival, and for treatment planning in patients with hepatocellular carcinoma (HCC) treated with 90Y-loaded glass microspheres (TheraSphere®).MethodsTheraSphere® was administered to 71 patients with inoperable HCC. MAA SPECT/CT quantitative analysis was used for the calculation of the tumour dose (TD), healthy injected liver dose (HILD), and total injected liver dose. Response was evaluated at 3 months using EASL criteria. Time to progression (TTP) and overall survival (OS) were evaluated using the Kaplan-Meier method. Factors potentially associated with liver toxicity were combined to construct a liver toxicity score (LTS).ResultsThe response rate was 78.8 %. Median TD were 342 Gy for responding lesions and 191 Gy for nonresponding lesions (p < 0.001). With a threshold TD of 205 Gy, MAA SPECT/CT predicted response with a sensitivity of 100 % and overall accuracy of 90 %. Based on TD and HILD, 17 patients underwent treatment intensification resulting in a good response rate (76.4 %), without increased grade III liver toxicity. The median TTP and OS were 5.5 months (2–9.5 months) and 11.5 months (2–31 months), respectively, in patients with TD <205 Gy and 13 months (10–16 months) and 23.2 months (17.5–28.5 months), respectively, in those with TD >205 Gy (p = 0.0015 and not significant). Among patients with portal vein thrombosis (PVT) (n = 33), the median TTP and OS were 4.5 months (2–7 months) and 5 months (2–8 months), respectively, in patients with TD <205 Gy and 10 months (6–15.2 months) and 21.5 months (12–28.5 months), respectively, in those with TD >205 Gy (p = 0.039 and 0.005). The median OS was 24.5 months (18–28.5 months) in PVT patients with TD >205 Gy and good PVT targeting on MAA SPECT/CT. The LTS was able to detect severe liver toxicity (n = 6) with a sensitivity of 83 % and overall accuracy of 97 %.ConclusionDosimetry based on MAA SPECT/CT was able to accurately predict response and survival in patients treated with glass microspheres. This method can be used to adapt the injected activity without increasing liver toxicity, thus defining a new concept of boosted selective internal radiation therapy (B-SIRT). This new concept and LTS enable fully personalized treatment planning with glass microspheres to be achieved.

Hepatocellular carcinomas (HCCs) exhibit a diversity of molecular phenotypes, raising major challenges in clinical management. HCCs detected by surveillance programs at an early stage are candidates for potentially curative therapies (local ablation, resection, or transplantation). In the long term, transplantation provides the lowest recurrence rates. Treatment allocation is based on tumor number, size, vascular invasion, performance status, functional liver reserve, and the prediction of early (<2 years) recurrence, which reflects the intrinsic aggressiveness of the tumor. Well‐differentiated, potentially low‐aggressiveness tumors form the heterogeneous molecular class of nonproliferative HCCs, characterized by an approximate 50% β‐catenin mutation rate. To define the clinical, pathological, and molecular features and the outcome of nonproliferative HCCs, we constructed a 1,133‐HCC transcriptomic metadata set and validated findings in a publically available 210‐HCC RNA sequencing set. We show that nonproliferative HCCs preserve the zonation program that distributes metabolic functions along the portocentral axis in normal liver. More precisely, we identified two well‐differentiated, nonproliferation subclasses, namely periportal‐type (wild‐type β‐catenin) and perivenous‐type (mutant β‐catenin), which expressed negatively correlated gene networks. The new periportal‐type subclass represented 29% of all HCCs; expressed a hepatocyte nuclear factor 4A–driven gene network, which was down‐regulated in mouse hepatocyte nuclear factor 4A knockout mice; were early‐stage tumors by Barcelona Clinic Liver Cancer, Cancer of the Liver Italian Program, and tumor–node–metastasis staging systems; had no macrovascular invasion; and showed the lowest metastasis‐specific gene expression levels and TP53 mutation rates. Also, we identified an eight‐gene periportal‐type HCC signature, which was independently associated with the highest 2‐year recurrence‐free survival by multivariate analyses in two independent cohorts of 247 and 210 patients. Conclusion: Well‐differentiated HCCs display mutually exclusive periportal or perivenous zonation programs. Among all HCCs, periportal‐type tumors have the lowest intrinsic potential for early recurrence after curative resection. (Hepatology 2017;66:1502–1518).

Intrahepatic cholangiocarcinoma (ICC) is the second most common type of primary cancer in the liver. ICC is an aggressive cancer with poor prognosis and limited therapeutic strategies. The identification of new drug targets and prognostic biomarkers is an important clinical challenge for ICC. The presence of an abundant stroma is a histological hallmark of ICC. Given the well-established role of the stromal compartment in the progression of cancer diseases, we hypothesized that relevant biomarkers could be identified by analyzing the stroma of ICC. By combining laser capture microdissection and gene expression profiling, we demonstrate that ICC stromal cells exhibit dramatic genomic changes. We identified a signature of 1,073 nonredundant genes that significantly discriminate the tumor stroma from nontumor fibrous tissue. Functional analysis of differentially expressed genes demonstrated that up-regulated genes in the stroma of ICC were related to cell cycle, extracellular matrix, and transforming growth factor beta (TGFb) pathways. Tissue microarray analysis using an independent cohort of 40 ICC patients validated at a protein level the increased expression of collagen 4A1/COL4A1, laminin gamma 2/LAMC2, osteopontin/SPP1, KIAA0101, and TGFb2 genes in the stroma of ICC. Statistical analysis of clinical and pathological features demonstrated that the expression of osteopontin, TGFb2, and laminin in the stroma of ICC was significantly correlated with overall patient survival. More important, multivariate analysis demonstrated that the stromal expression of osteopontin was an independent prognostic marker for overall and disease-free survival. Conclusion: The study identifies clinically relevant genomic alterations in the stroma of ICC, including candidate biomarkers for prognosis, supporting the idea that tumor stroma is an important factor for ICC onset and progression. (HEPATOLOGY 2013;58:1992-2000 I ntrahepatic cholangiocarcinomas (ICC) account for 5%-10% of liver primary cancers. 1 ICC usually arise from epithelial cells of the intrahepatic small bile ducts, although a recent report in mice suggested that ICC might also originate from the conversion of mature hepatocytes. 2 Over the last decade, the incidence of ICC has increased significantly in Western countries. 3,4 This trend could be related to a better histological diagnosis of ICC and/or to the rising incidence of the main risk factors for ICC: cholangitis, chronic hepatitis B and C, diabetes, and obesity. 5 Liver resection remains the only curative treatment for ICC but is associated with a high rate of recurrence. 6 Recently, we showed that hilar lymph node metastasis,